Mechanical stability of αT-catenin and its activation by force for vinculin binding

Figures

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  FIGURE 1:. Protein constructs and experimental design. (A) Domain organization of αE-catenin and αT-catenin. The VBS from amino acid 300–360 is indicated. (B) Protein constructs for single-molecule stretching experiments used in this study. (C) Schematic of the M123 construct tethered between a coverslip and a superparamagnetic bead under an applied force F.

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  FIGURE 2:. Force-response of αT-catenin middle domains. Representative changes of the height of the bead tethered to an M123 construct (A) and a 2M1 construct (B) when subjected to a linearly increasing force loading rate of 1.0 ± 0.1 pN/s. The arrows indicate the domain unfolding events accompanied by a stepwise height increase. (C, D) The unfolding force distribution obtained from the M123 construct (C) and the 2M1 construct (D). The vertical dashed lines indicate the boundary between different mechanical stability groups indicated by I, IIa, and IIb. The black line in C is three-peak Gaussian fitting to the unfolding force histogram. The colored lines are Gaussian distributions to individual groups plotted based on the fitted parameters. The line in D is the single-peak Gaussian fitting to the unfolding force histogram of the 2M1 construct.

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  FIGURE 3:. Mechanical stability of the M1 domain. (A) Representative time traces of the height of the bead tethered to a 2M1 construct at various constant forces over a duration of 60 s and smoothed over 0.1 s (black). The digitized stepwise changes in bead height were determined based on hidden Markov analysis using stepfit1 software (red) (Aggarwal et al., 2012). (B) The force-dependent unfolding (black) and folding (red) rates of the M1 domain calculated from the dwell times of the folded/unfolded states obtained from A. The errors are estimated using Bootstrap analysis (Supplemental Methods). The rates are fitted with Bell’s model where the best fitting parameters are indicated in the figure panel.

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  FIGURE 4:. Force-dependent VD1 binding. (A) Representative changes in the height of the bead tethered to a M123 construct when subjected to a linearly increasing (3.5 ± 0.4 pN/s) force loading rate before (gray traces) and after the addition of 50 nM VD1 (colored traces), smoothed over 0.1 s. In the presence of VD1, the bead height is higher than that before introducing VD1 at low forces (dashed box), indicating VD1 binding inhibits M1 refolding. At forces >20 pN, there is an additional ∼3- to 4-nm step indicating the unbinding of VD1 from M1 (insert, arrow showing two of the steps). (B) Four representative time traces of the height change of a bead tethered to an originally folded M123 construct in 50 nM VD1 held at a constant force of ∼2 pN for 1000 s (before the break) and those of the same tethers for another 1000 s (after the break) at the same force after unfolding the M1 domain by transiently holding the tethers at ∼8 pN for 2 min. (C) A representative time trace of the height of a bead tethered to a M123 construct during force jumping between ∼8 pN and ∼31 pN. The two 3- to 4-nm stepwise bead height increases at ∼31 pN indicate VD1 dissociation events (black arrows). (D) The time…